1. Technical Field of the Invention
The present invention relates to a method for detecting multiple different chromosome regions or DNA regions in a cell for detection of structural chromosome aberrations, wherein the chromosome aberrations have at least two breaking point regions within a chromosome, based on directly or indirectly labeled nucleic acid fragments (probes) and a preparation suitable for this method.
2. Description of the Related Art
Many malignancies are based on structural chromosome mutations, such as translocations, inversions and segmental duplications. These changes are usually detected as predictive, prognostic or differential diagnostic markers by in situ hybridization (ISH) using fluorescence-labeled (fluorescence ISH (FISH)) nucleic acid fragments, so-called probes or hapten-labeled probes, which are then detected with antibodies and visualized by color reactions under light microscopy (bright field ISH (BrISH)—also including chromogenic ISH (CISH) and silver-enhanced ISH (SISH)).
The advantage of FISH here is that multiple genomic regions can be detected simultaneously but still differentiated clearly from one another without increased effort in performing the method. To do so, nucleic acid fragments, which address different genomic regions, are labeled with, i.e., coupled to, different fluorescent dyes, which differ from one another in their absorption spectrum and/or emissions spectrum. When such multicolor probes are used, e.g., on metaphase chromosome preparations or on interphase cell nucleus preparations, the individual colors can be represented separately from one another by using specific microscope filter sets, which conduct precisely defined wavelength ranges of light for excitation of dyes onto the preparation and allow precisely defined wavelength ranges of light emitted by the dyes to pass through to the analyzer (so-called single-bandpass filter set), or the different fluorescent signals of multiple fragments are represented at the same time (in the case of two different fluorescent dyes, we speak here of a dual-bandpass filter set).
However, there are definite limits to simultaneous visualization because the absorption ranges and emission ranges of the dyes are often so close to one another that they cannot be separated from one another by the microscope filter sets. Furthermore, two superimposed signals of different colors result in the perception of mixed colors (e.g., yielding red (or orange) and green and thereby yellow), which cannot be differentiated from signals of the same color, which are the result of another fluorochrome and are not formed by overlapping. For these reasons, usually only two colors (orange/red and green) or three colors (orange/red and green simultaneously with a blue nuclear counterstain (DAPI)) are observed simultaneously in routine FISH. Approximately the same restrictions as those that can be visualized for FISH also apply to BrISH, where the state of the art is to use two haptens, usually selected from the group of biotin, dinitrophenyl (DNP) and digoxigenin, and two antibody-coupled enzymes, usually alkaline phosphatase and peroxidase (Carbone et al., 2008; Hopman et al., 1997; Laakso et al., 2006; Mayr et al., 2009; Tanner et al., 2000).
These restrictions in simultaneous visualization have a definitive influence on the compositions of the probes for detection of translocations and inversions. There are in principle two definitive techniques and fundamental probe compositions here: the principle of the formation of fusion signals (so-called dual-color-dual-fusion approaches) (WO 02093130, Dewald et al., 1998; Dewald et al., 2000; Wan et al., 2003) and/or of the separation of fusion signals (so-called dual-color-break-apart or dual-color-split approaches) (van der Burg et al., 1999; Boomer et al., 2001; van der Burg et al., 2004). In the following discussion of these two principles and derived signal patterns, it should be noted that a normal cell is usually diploid, i.e., each allele is present twice. Since usually only one of the two alleles is affected by aberrations, then the normal signal of the allele not affected by the aberration will usually be visible in addition to the aberrant signal. For a better understanding, the signal pattern of the normal signal is not always described explicitly below.
In dual-color-dual-fusion approaches, the region of breaking point 1 is flanked proximally and distally by nucleic acid fragments of the same color (e.g., orange), while the region of the breaking point 2, i.e., the reciprocal translocation partner, is flanked proximally and distally by nucleic acid fragments of a second color (e.g., green). The normal situation, i.e., without chromosomal breaks in the region of the two translocation partners is characterized here by a green signal and by a spatially separate orange signal.
In the case of a reciprocal translation, there are breaks within the breaking points of the two translocation partners, and the proximal region of the one translocation partner is fused to the distal region of the other partner and vice-versa. This therefore results in two green/orange signal pairs, also known as fusion signals, because the different colored signals often overlap, so a mixed color signal is visible. The disadvantage of these probe techniques is that the fusion signals are formed only when the breaking points of the two translocation partners are situated in the region of the respective labeled nucleic acid fragments. In translocations affecting only one of the two partners, no fusion signals are formed. This results only in the creation of an additional signal having the color of the signal which is characteristic of the partner affected by the translocation. In other words, an additional green signal, for example, is formed when the breaking point of the translocation is in the region covered by the nucleic acids labeled with green fluorochrome. However, such additional signals may often be covered by other cell nuclei, which may be situated close to one of the other signals of the same color and thus cannot be perceived as an additional signal or are lost due to tissue section artifacts. In this case, there may be a misdiagnosis because a translocation affecting only the one translocation partner is not perceived.
In dual-color-dual-fusion approaches, the region of breaking point 1 is flanked proximally and distally by nucleic acid fragments of the same color (e.g., orange), while the region of the breaking point 2, i.e., the reciprocal translocation partner, is flanked proximally and distally by nucleic acid fragments of a second color (e.g., green). The normal situation, i.e., without chromosomal breaks in the region of the two translocation partners, is characterized here by a green signal and by a spatially separate orange signal.
The disadvantage of these probe compositions is that it is impossible to obtain information about the translocation partner involved. Furthermore, due to the sighting axis with which the observer views the interphase cell nucleus, for example, it may happen that two spatially separate signals, which are situated one above the other with respect to the viewing axis, are still perceived as fusion signals.
In addition to the two-color applications mentioned above, three-color, four-color and five-color probes are also used in FISH analyses. In addition to the much stronger standard fluorescent colors orange, red and green, the other weaker colors that are available, e.g., gold or red or blue, are also used. Therefore, in routine applications, for example, multicolor FISH probes are used only for detection of deletions or amplifications (EP 1035215 B1, WO 2007/028031, EP 0549709 B1) because repetitive sequences can usually be accessed here in the labelings for amplification of the color intensities, e.g., blue and gold.
The triple FISH approaches described by Finkel et al. (2009) address the detection of different translocation events, which may cluster side-by-side in a chromosomal region (i.e., different genes located in proximity are affected). For the corresponding analysis of the signal patterns, only two colors which detect a single aberration are used, wherein the third color then does not play a role.
With respect to BrISH using more than two colors, Hopman et al. (1997) describe how a chromogenic triple in situ hybridization is performed, aimed in general at detecting three repetitive chromosomal regions. According to the state of the art today, translocations are detected by means of BrISH only using two haptens and thus two colors.
Also unknown is the use of the BrISH method for detection of inversions and insertions.
Inversions and in particular small inversions cannot be detected with the techniques and methods described in the patent applications WO 02093130 A3 and WO 2005/111235 A2. WO 2005/111235 A2 describes a method involving the use of three color probes. However, the chromosomal region labeled by the third label of a probe is not affected directly by a change so that in a change in chromosome structure, the first fusion signal is eliminated, resulting in a new split signal and a new fusion signal. It is difficult or impossible to distinguish this event from a normal situation in the case of inversions, in particular small inversions.
WO 02093130 A3 discloses a method for detection of chromosomal translocations using two or four labels/dyes, wherein the probes are hybridized for different chromosomes. This method is suitable for detection of inversions only to a limited extent. Furthermore the analytical method is much more complex due to the plurality of signals that can be derived from four labels/dyes.
All the methods and probes compositions currently available for BrISH and FISH in conjunction with inversions, in particular but not limited to tumors, have shortcomings. There are no probe compositions and methods and their resulting signal patterns which allow reliable detection of an inversion in all cases. This aberration event is not usually detected in the case of small inversions in particular, i.e., inversions of short genomic segments (for example, regions of less than 20 Mb).
The object of the present invention is therefore to provide a method with which reliable detection of structural chromosome aberrations is made possible, wherein the chromosome aberrations have at least two breaking point regions within a chromosome.